INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION TOXICOLOGICAL EVALUATION OF SOME FOOD COLOURS, EMULSIFIERS, STABILIZERS, ANTI-CAKING AGENTS AND CERTAIN OTHER SUBSTANCES FAO Nutrition Meetings Report Series No. 46A WHO/FOOD ADD/70.36 The content of this document is the result of the deliberations of the Joint FAO/WHO Expert Committee on Food Additives which met in Rome, 27 May - 4 June 19691 Food and Agriculture Organization of the United Nations World Health Organization 1 Thirteenth report of the Joint FAO/WHO Expert Committee on Food Additives, FAO Nutrition Meetings Report Series, in press; Wld Hlth Org. techn. Rep. Ser., in press. BRILLIANT BLUE FCF Biological Data Biochemical aspects The colour was administered orally to rats as a two per cent. aqueous solution at a level of 200 mg per rat. Almost the entire amount was excreted unchanged in the faeces within 40 h after administration. In a later investigation, the presence of the colour in the bile was observed in rats, rabbits and dogs after oral administration. In the case of the dog, the amount did not exceed five per cent. of the dose administered (Hess & Fitzhugh, 1953; 1954; 1955). Administration of an aqueous solution of the colour by stomach tube resulted in 89 per cent. excretion in the faeces; none was found in the urine. After subcutaneous injection of 80-100 mg some 79 per cent. was excreted; 77 per cent. appeared in the faeces and 2.5 per cent. in the urine (Imperial Chemical Industries, 1958). Acute toxicity Animal Route LD50 Reference mg/kg body weight Mouse Subcutaneous 4 600 Gross, 1961 Rat Oral > 2 000 Lu & Lavallée, 1964 Groups of five young rats were given injections of the colour subcutaneously twice daily for three days. The rats were killed on the fourth day. The colour was administered in aqueous solution at a level of 250 mg/kg body weight each injection. No oestrogenic activity was detected. No other abnormalities were found (Graham & Allmark, 1959), Special studies o-Sulfobenzaldehyde, one of the components of Brilliant Blue FCF, was fed to groups of three-week-old rats, 10 males and 10 females per group, at levels of 0, 0.25, 0.5, 1 and 2 per cent. for a period of 13 weeks followed by gross and histopathology examinations. The "no-effect" level of o-sulfobenzaldehyde was considered to be two per cent. (United States Food and Drug Administration, 1969). Short-term studies Mouse: Daily injections of 2 mg of colour over 30 days were well tolerated by mice. Some animals showed swelling of the liver and spleen after receiving a total of 4 mg (Gross, 1961). Mice fed 1200 mg of the dye ever 19 days showed no damage (Gross, 1961). Rat: Subcutaneous injection of 1 ml of 0.8 per cent. aqueous solution twice weekly produced histological changes suggestive of subsequent sarcoma formation unassociated with chemical carcinogenic potential (Grasso & Golberg, 1966). Dog: Studies were conducted on beagle dogs fed diets containing one per cent. and two per cent. of the colour for one year. No gross or microscopic changes due to treatment were seen (Hansen et al., 1966). Cat: Parenteral injection into cats of a five per cent. aqueous solution, 1.0 g on the first day and 0.1 g from the ninth to the eighteenth day, did not produce methaemoglobinaemia or Heinz bodies (Gross, 1965). Mouse: A group of 57 male and 43 female mice were given 1 mg colour per day in the diet. Observations extended over 500-700 days. No evidence of carcinogenic action was found (Waterman & Lignac, 1958). Subcutaneous injections of 10 doses of 4 mg followed by 50 doses of 6 mg showed no tumour production after 78 weeks (Imperial Chemical Industries, 1962). Rat: The colour was fed in a concentration of four per cent. of the diet to five male and five female rats for 600 days. Gross staining of the glandular stomach and some granular deposits in the stomach but no tumours were observed (Willheim & Ivy, 1953). Eighty-five rats were fed a diet containing 0.1 per cent. of the colour for their life span. The daily intake was 10-15 mg. No tumours were found (Klinke, 1955). Groups of 24 weanling Osborne-Mendel rats, evenly divided by sex, were fed the colour at levels of 0, 0.5 per cent., 1.0 per cent., 2.0 per cent. and 5.0 per cent. for two years. No outward effects were observed during the experiment. Gross and microscopic examination revealed only incidental lesions, unrelated to treatment, with no effect on tumour production. The statistical evaluation revealed no changes in organ weights, mortality, or growth. Haematology was normal (Hansen et al., 1964). Four groups of 15 male and 15 female rats were given diets containing, 0, 0.03 per cent., 0.3 per cent. and 3.0 per cent. of the colour for 75 weeks. No depression of growth, food consumption or food efficiency was found. Mortality was increased in the three per cent. group due to respiratory infection unrelated to the administration of the compound. Haematological findings were normal (Mannell et al., 1962). Subcutaneous injection of various preparations of the colour have been reported to produce fibrosarcomas at the site of the injection (Nelson & Hagan. 1953; Gross, 1961; Hansen et al., 1966). However, no tumours were produced by a course of subcutaneous injections of 0.5 ml of a four per cent. solution in isotonic saline (Mannell & Grice, 1964). Eighty-four male and 84 female Wistar rats were given twice-weekly 10 doses of 20 mg per rat as a two per cent. solution followed by 50 doses of 30g per rat as a six per cent, solution. One hundred and nineteen of the 168 rats developed sarcomas at the site of injection. Only seven animals survived two years. Males had no other tumours but females had six mammary, one ovarian, one uterine tumour and two hepatomas. Twenty-six controls survived two years out of 48 and the females developed one mammary, one ovarian and four uterine tumours (Imperial Chemical Industries, 1962). Six male and six female Slonacker rats were treated the same way as Wistar rats. All died within 420 days, tumours developing at the injection site in five males and three females. Out of 12 controls, 10 were alive at 400 days, No tumours developed in controls (Imperial Chemical Industries, 1962). Comments The production of a high percentage of local sarcomata at the site of subcutaneous injection in rats has led in the past to considerable discussion and consequently to extensive studies on this colour. The production of these sarcomata is considered to be related to the physico-chemical properties of the colour and special condition of the experiment and does not constitute evidence of carcinogenicity by the oral route. Biochemical studies have shown that the colour is poorly absorbed and is almost completely excreted in the faeces after parenteral administration. Extensive long-term studies in two species are available. In addition a 13-week study in rats with o-sulfobenzaldehyde, one of the components of commercial Brilliant Blue FCF, has been carried out. Oral feeding produced no pathological changes at the highest levels used in adequate experiments. Biochemical studies on metabolism using modern techniques are desirable. EVALUATION Level causing no toxicological effect in the rat Five per cent, in the diet (= 50 000 ppm) equivalent to 2500 mg/kg body weight/day. Estimate of acceptable daily intake for man mg/kg body weight/day Unconditional acceptance 0-12.5 REFERENCES Graham, R. C. B. & Allmark, M. G. (1959) Toxic. Appl.Pharmac., 1, 144 Grasso, P. & GoIberg L. (1966) Fd. Cosmet. Toxicol., 4, 269 Gross, E. (1961) Z. Krebsforsch., 64, 287 Hansen, W. H. et al. (1966) Toxicol. Appl. Pharmacol., 8, 29 Hess, S. M. & Fitzhugh, O. G. (1953) Fed. Proc., 12, 330 Hess, S. M. & Fitzhugh, O. G. (1954) Fed. Proc., 13, 365 Hess, S. M. & Fitzhugh, O. G. (1955) J. Pharmacol. exp. Ther., 114, 38 Imperial Chemical Industries (1958) Unpublished report Imperial Chemical Industries (1962) Unpublished report Lu, F. C. & Lavallée., A. (1964) Canad. pharm. J., 97, 30 Mannell, W. A., Grice, H. C. & Allmark, M. G. (1962) J. Pharm. Pharmacol., 14, 378 Mannell, W. A. & Grice, H. C. (1964) J. Pharm. Pharmacol., 16, 56 Nelson, A. A. & Hagan, E. C. (1953) Fed. Proc., 12, 397 United States Food and Drug Administration (1969) Unpublished report Willheim, R. & Ivy, A. C. (1953) Gastroenterology, 23, 1 Waterman N. & Lignac, G. O. E. (1958) Acta. physiol. pharmacol. neerl., 7, 35
See Also: Toxicological Abbreviations BRILLIANT BLUE FCF (JECFA Evaluation)